Targeting RAS guanyl releasing protein 1 promotes T lymphocytes infiltrations and improves anti‐programmed death receptor ligand 1 therapy response of triple‐negative breast cancer

Dear Editor, Triple-negative breast cancer (TNBC) is characterized as an aggressive form of breast cancer (BC).1,2 Targeting immune checkpoint inhibitors (ICIs), such as programmed death receptor ligand 1 (PD-L1), has been proposed as a potential strategy for TNBC treatment.3 Several monoclonal antibodies targeting PD-L1, such as Atezolizumab, have been approved to be used for TNBC treatment.4 Despite ICIs show long-term treatment effects on a faction of TNBCpatients, the decreased response rates still widely exist.5 The intrinsic and extrinsic microenvironment heterogeneity of TNBC, including insufficient infiltration of cytotoxic T lymphocytes and immunosuppressive tumour microenvironment (TME), might drive therapeutic resistance of ICIs.6,7 Hence, novel targets that can enhance the immunotherapeutic efficacy of TNBC need to be identified. In the present work, we extracted three Gene Expression Omnibus (GEO) datasets regarding positive or negative PD-L1 states in TNBC (GSE100824, GSE107764 and GSE157284). The comparisons were conducted with the cutoff of p < .05 and Foldchange > 1.5 (Table S1), and only one co-upregulated gene (RAS guanyl releasing protein 1, RASGRP1) was identified, whereas no co-downregulated geneswere found (Figure 1A,B andFigure S1A–C). Validating by Spearman correlation analysis, RASGRP1 expression was positively correlated with PD-L1 expression in GSE88847, GSE180775, and GSE58812 (Figure 1C–E). Further investigations were conducted on the clinical significance of RASGRP1 in TNBC. Through BEST database and Xiantao Tool, significantly decreased RASGRP1 expression was demonstrated in ERand PR-negative groups, as well as advanced TNM stages of BC samples (Figure S1D–I). The TISCH database further revealed that decreased RAS-


Targeting RAS guanyl releasing protein 1 promotes T lymphocytes infiltrations and improves anti-programmed death receptor ligand 1 therapy response of triple-negative breast cancer
Dear Editor, Triple-negative breast cancer (TNBC) is characterized as an aggressive form of breast cancer (BC). 1,2 Targeting immune checkpoint inhibitors (ICIs), such as programmed death receptor ligand 1 (PD-L1), has been proposed as a potential strategy for TNBC treatment. 3 Several monoclonal antibodies targeting PD-L1, such as Atezolizumab, have been approved to be used for TNBC treatment. 4 Despite ICIs show long-term treatment effects on a faction of TNBC patients, the decreased response rates still widely exist. 5 The intrinsic and extrinsic microenvironment heterogeneity of TNBC, including insufficient infiltration of cytotoxic T lymphocytes and immunosuppressive tumour microenvironment (TME), might drive therapeutic resistance of ICIs. 6,7 Hence, novel targets that can enhance the immunotherapeutic efficacy of TNBC need to be identified.
In the present work, we extracted three Gene Expression Omnibus (GEO) datasets regarding positive or negative PD-L1 states in TNBC (GSE100824, GSE107764 and GSE157284). The comparisons were conducted with the cutoff of p < .05 and Foldchange > 1.5 (Table S1), and only one co-upregulated gene (RAS guanyl releasing protein 1, RASGRP1) was identified, whereas no co-downregulated genes were found ( Figure 1A,B and Figure S1A-C). Validating by Spearman correlation analysis, RASGRP1 expression was positively correlated with PD-L1 expression in GSE88847, GSE180775, and GSE58812 ( Figure 1C-E). Further investigations were conducted on the clinical significance of RASGRP1 in TNBC. Through BEST database and Xiantao Tool, significantly decreased RASGRP1 expression was demonstrated in ER-and PR-negative groups, as well as advanced TNM stages of BC samples ( Figure S1D-I).
The TISCH database further revealed that decreased RAS-Xi Chen and Yuanliang Yan contributed equally to this work.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. GRP1 in malignancy cells compared to immune cells of BC samples ( Figure 1F). Through Kaplan-Meier Plotter, BC or TNBC patients with lower expression of RASGRP1, underwent unfavorable distant metastasis-free survival, overall survival and recurrence-free survival ( Figure 1G-I and Figure S1J-L). The nomogram model further revealed that RASGRP1 could function as effective predictor of 3-, 5-and 10-year survivals in BC patients ( Figure 1J). The univariate and multivariate COX analysis also confirmed that lower expression of RASGRP1 was an independent risk factor of survivals in BC patients (Table S2).
To explore its biological effects, KEGG enrichment analysis revealed that RASGRP1 might be involved in the cell adhesion molecules, focal adhesion, and pathways in cancer ( Figure 2A). Hence, in vitro analysis was further implemented to explore its effect. We overexpressed RASGRP1 in two human TNBC cell lines, BT549 and MDA-MB-231 ( Figure 2B). As shown in Figure 2C-J, overexpression of RASGRP1 significantly inhibited cell viability, invasion and migration in in vitro. Furthermore, the GO and GSEA analysis manifested those genes enriched with RASGRP1 mainly involved in the immunity-related terms, such as immune system process and T cell activation in BC ( Figure 2K and Figure S2A-D). Through hallmark gene-sets analysis, enriched pathways of RASGRP1 were associated with TNF-α signalling in CD8 + T cells in GSE114727_10× ( Figure 2L,M) and GSE114727_inDrop ( Figure 2N) in TISCH database. As a kind of cytokine, TNF-α was reported to involve in various biological effects and promote cell proliferation, tumour recurrence, and metastasis in TNBC. 8,9 We then examined the levels of TNF-α in vitro. Upregulation of RASGRP1 was verified to reduce TNF-α in human BT549 and MDA-MB-231 cells ( Figure 2O,P). Similarly, stable overexpression of To validate its role in TME, the BEST database and Xiantao tool showed that RASGRP1 was positively correlated with immune infiltration score and T-lymphocyte infiltrations in BC patients ( Figure 3A and Figure S2E,F). In a TNBC dataset (GSE55812), expression of RASGRP1 was positively related to CD8 + T cells, central memory CD8 + T cells, and effector memory CD8 + T cells ( Figure 3B-E). Expression of RASGRP1 displayed positive associations with a series of T-lymphocyte-related immunomarkers, particularly CD8A, based on TNBC datasets ( Figure 3E,F, and Figure S2G-I). Moreover, IHC staining of TNBC samples revealed that the intensity of RASGRP1 in malignancy was positively correlated with CD8 in the stroma ( Figure 3G,H). Through datasets from the TISCH database, single-cell RNA-seq (sc-RNAseq) analysis was further performed to verify the relationship between RASGRP1 and CD8 + T cell infiltrations. The sc-RNAseq analysis showed that RASGRP1 were mainly distributed in CD8 + T cells surrounding TME of BC ( Figure 3I-K and Figure S3A-Q). Additionally, sc-RNAseq analysis was conducted to testify to the expression of RASGRP1 in TNBC samples based on EMTAB8107 and GSE136206. As shown in Figure 3L-T, samples with higher expression of RASGRP1 in malignant cells, showed a  relatively higher abundance of CD8 + T cells in stroma cells. These data suggested that RASGRP1 might be relevant to the infiltrations of CD8 + T cells in TNBC.
We further investigated whether RASGRP1 could affect anti-PD-L1 response in TNBC. Through the TIDE algo-rithm, the scores predicted unfavourable ICI responses were decreased in the high RASGRP1 group ( Figure S4). Subsequently, an orthotopic transplantation in vivo mice model was conducted to confirm its function. The BALB/c mice, bearing xenograft tumours of 4T1 cells with vector or stably overexpressed RASGRP1, were treated with PBS or anti-PD-L1 agent (Atezolizumab) ( Figure 4A). As shown in Figure 4B-E, overexpression of RASGRP1 led to lower tumour weight and volume, while no change was found in body weight. The above results suggested that RAS-GRP1 in tumour cells may be correlated with infiltrations of CD8 + T cells, which act as a predominant factor for ICI responses. 10 Hence, we explored the proportion of CD8 + T cells from peripheral blood in each group. As expected, in anti-PD-L1 treated groups, RASGRP1-overexpression showed higher levels of CD8 + T cells, while no significant difference was found in PBS-treated groups ( Figure 4F-G). Above results indicated that upregulation of RASGRP1 could enhance the anti-PD-L1 response in TNBC.
In all, our study sheds light on the correlation of RAS-GRP1 expression in tumour cells and infiltrations of CD8 + T cells in TNBC TME ( Figure 4H). The methods utilized in this work are comprised in Table S3. Previous studies have demonstrated that RASGRP1 involves in mediating immune responses in various diseases. For instance, loss-of-function mutations in RASGRP1 impede T-cell activation and proliferation by interruption of receptor signalling, resulting in lymphoma-or leukemogenesis. 11 In hepatocellular carcinoma, overexpression of RASGRP1 inhibits the inflammation-associated cancer development, by reducing the production of the proinflammatory cytokine IL-6. 12 From now on, the nature of RASGRP1 in TME of TNBC is still blurry. Considering the negative regulation between RASGRP1 and TNF-α signalling, this effect might be caused by the release of specific cytokines in tumour cells. Our finding suggested that RASGRP1 predicted poor anti-PD-L1 agent response and might work as a promising immunotherapy target for anti-PD-L1 therapy in TNBC.

A C K N O W L E D G E M E N T S
This study is supported by grants from the National Natural Science Foundation of China (82272659), the Science and Technology Innovation Program of Hunan Province (2021RC3029; 2022RC1210) and the China Postdoctoral Science Foundation (2021T140754, 2020M672521).